Andrographis paniculata as a Next-Generation Hepatoprotective Agent: An Updated Review on Andrographolide-Mediated Molecular Mechanisms and Translational Applications in Liver Disorders

1.1. Global Burden and Clinical Significance of Liver Disorders

Liver disorders pose a serious public health problem due to their growing prevalence, clinical progression and significant burden in terms of morbidity and early death. The liver plays an important metabolic role in the body and functions to metabolize nutrients, detoxify substances, synthesize proteins, make bile, regulate immunity, and maintain homeostasis throughout the body. Therefore, even a slight damage of the liver tissue can give rise to many metabolic and physiological imbalances [1]. Liver diseases include metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), alcohol-associated liver disease, viral hepatitis, drug-induced liver disease, liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). While the etiologies of these diseases differ, multiple overlapping pathogenic pathways, such as oxidative stress, chronic inflammation, mitochondrial dysfunction, extracellular matrix deposition, and hepatocellular apoptosis, play a role in their initiation and progression. Chronic liver disease has become a significant public health problem worldwide, due to the gradual shift of dietary habits, sedentary lifestyle, obesity, type 2 diabetes mellitus, heavy drinking, and chronic viral infections. In contemporary epidemiological reviews, MASLD has been identified as the most common type of chronic liver disease, impacting one-third of the adult population worldwide. While prevalence estimates differ based on diagnostic criteria and population characteristics, the Global Burden of Disease (GBD)-based analysis estimated that there were around 1.27 billion prevalent cases of MASLD in the world in 2021. As metabolic disorders become more prevalent, liver complications associated with MASLD will increasingly place a burden on health care systems. Chronic hepatitis B and C infections continue to be significant causes of advanced liver disease, especially in areas where screening, vaccination, and antiviral therapy are not widely available. Likewise, chronic alcohol consumption leads to liver lipid accumulation, oxidative stress, inflammation and progressive fibrosis [2]. Beyond liver injury, these disorders are clinically significant due to associated portal hypertension, ascites, hepatic encephalopathy, coagulation abnormalities and multiorgan complications of severe liver dysfunction. Furthermore, chronic liver damage could progress to cirrhosis and HCC, significantly decreasing survival and quality of life. The fact that many liver diseases are chronic and have a long latent period often makes it difficult to detect the disease early and to treat it appropriately. The progressive hepatic injury at a molecular level is due to the excessive production of Reactive Oxygen Species (ROS), defective mechanisms of antioxidant response, activation of inflammatory transcription factors and failure of programmed cell death. These processes work in concert with metabolic abnormalities and fibrogenic signals to create a vicious cycle of inflammation and tissue remodeling. Thus, compounds that can modulate all these processes (oxidative stress, inflammatory mediators, metabolic abnormalities, and fibrotic responses) are of high interest for therapeutic use. Increases in disease burden illustrate the need for effective, safe and mechanistically targeted hepatoprotective interventions [3].

1.2. Limitations of Conventional Hepatoprotective Therapies

The treatment of liver disorders is mainly based on the pharmacologic treatment of the disease, modification of lifestyle, control of underlying metabolic disorders and supportive clinical management. Chronic hepatitis B and C now have better management with the use of antiviral agents, and targeted use of steroids, immunosuppressive drugs, etc. is used in selected inflammatory and autoimmune liver diseases. Diet, weight loss, exercise and avoidance of alcohol are still key parts of treating metabolic and alcohol induced liver diseases. However, the effectiveness of treatment often also relies on the stage of the disease, patients' characteristics, any underlying conditions and on long-term compliance [4]. Among the drawbacks of the traditional treatments is the multifactorial and heterogeneous character of liver damage. There are several interventions currently available that target only a single etiological factor or molecular pathway, but not the entire network of oxidative, inflammatory, metabolic and fibrotic abnormalities that lead to disease progression. In addition, pharmacological treatment may be sub-optimal for those with advanced hepatic fibrosis and existing cirrhosis, especially with significant architectural remodeling. Patients with advanced liver disease are, therefore, likely to need complex multi-disciplinary management or liver transplantation. the treatment of metabolic liver disease has improved in recent years thanks to new therapeutic advances. For instance, in the U.S., resmetirom, a selective thyroid hormone receptor-β agonist was approved in March 2024 for certain adults with noncirrhotic MASH and moderate-to-advanced fibrosis. Clinical practice has been clarified since then in terms of patient selection, monitoring, and treatment of the disease. However, long-term clinical effects, treatment availability, side effects and relevance of different stages of the disease are still relevant. Likewise, the introduction of pharmacological innovations in the field of incretin-based therapy present other opportunities, which should be carefully assessed in the clinical setting before being used and have to be selected based on clinical presentation. A further problem is those that may arise in the process of treatment, such as drug interactions, adverse reactions and changes in the way that drugs are metabolized in those who have impaired liver function [5]. Drug exposure and risk for toxicity may be altered by progressive hepatic dysfunction, due to the critical role the liver plays in drug biotransformation. Furthermore, chronic treatment often involves extensive monitoring over long periods of time, which adds up in healthcare costs and can impact the adherence to treatment. Some diseases, such as late-stage cirrhosis and severe acute liver failure, do not have a lot of means to reverse established liver damage. These difficulties have led to the exploration of alternative therapeutic strategies which target several pathological processes. There has been growing scientific interest in natural bioactive compounds with antioxidant, anti-inflammatory, metabolic regulatory and antifibrotic properties. Their potential clinical value, however, should be established by extensive pharmacological characterization and toxicological studies and by carefully controlled clinical trials. Therapeutic safety and efficacy are not guaranteed by the natural origin. Therefore, the use of plant-derived compounds in hepatological treatment is considered investigational compounds or supplements until appropriate human studies have been performed and the evidence is established to allow their use in standard hepatological treatment [6].

1.3. Emerging Role of Medicinal Plants in Hepatoprotection

Medicinal plants are now emerging as a promising source for hepatoprotection. Medicinal plants are now gaining their importance in hepatoprotection. Throughout history, many medicinal plants have been used in traditional medicine to treat liver and stomach diseases, such as Ayurveda, traditional Chinese medicine, and other ethnomedicinal uses. The renewed scientific interest in herbal therapeutics has resulted from the development of new models of disease, analytical technologies, molecular pharmacology and phytochemistry [7]. The progress in these areas has allowed scientists to isolate particular bioactive components and to study their effect on intracellular signaling pathways involved in hepatocellular injury. While therapeutic interventions are targeted at a specific molecular target, some of the phytochemicals have several biological activities capable of affecting interrelated and potentially important mechanisms contributing to the progression of liver disease. A wide variety of secondary metabolites derived from plants have been shown in experimental models of liver injury to have various pharmacological activities, such as flavonoids, phenolics, terpenoids, alkaloids and glycosides. They suggest hepatoprotective mechanisms include scavenging of reactive species, enhancing endogenous antioxidant defense, reducing proinflammatory cytokine production, regulating lipid metabolism, maintaining mitochondrial integrity, and inhibiting hepatic stellate cell activation. Multiple phytochemicals have also been studied as modulators of transcription factors, signaling pathways, including nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear factor-κB (NF-κB), AMP-activated protein kinase (AMPK), and transforming growth factor-β (TGF-β). The relevance of these pathways is related to the close interplay between oxidative stress, inflammatory and metabolic dysfunction and fibrosis in the course of chronic hepatic injury. Some of the compounds which have been most studied, such as silymarin, curcumin, berberine, quercetin, and the different diterpenoids, have been found to have hepatoprotective properties. Experimental studies have indicated that some of these substances can enhance some of the biochemical markers of liver injury, diminish production of inflammatory mediators and diminish histopathologic changes [8]. However, promising results have not always been replicated in clinical practice. The complexity of various compositions of extracts, their bioavailability, the dosage applied, study design, and the models used for the diseases make the comparison difficult and the currently available evidence less generalizable. One of the main hurdles in the development of hepatoprotective agents from plants is its low aqueous solubility, high first-pass metabolism, low bioavailability and phytochemical variability. Moreover, certain herbal products may contain substances that are hepatotoxic or may be adulterated or contaminated with hepatotoxic components. Therefore, standardized extraction procedures, chemical fingerprinting, well-established analytical methods, and comprehensive safety evaluations are crucial for reproducible pharmaceutical development. In recent times, tremendous developments in the fields of nanotechnology, phytosomal formulations, lipid-based carriers and polymeric delivery systems have emerged, which provide possibilities to enhance the pharmaceutical performance of poorly soluble phytoconstituents. These methods could help improve the chemical stability of the drug, its absorption from the gastrointestinal tract, controlled release, and tissue distribution, but each must be evaluated separately for its therapeutic benefits. New research therefore is directed towards the development of novel drug candidates from pharmacologically active constituents of medicinal plants, which can then be chemically characterized and pharmacologically validated. In this context, much attention has been given to the Andrographis paniculata due to its rich phytochemical profile with diterpenoids and its experimentally reported biological activities relevant to the hepatoprotective effect [9,10].

1.4. Therapeutic Significance of Andrographis paniculata

Andrographis paniculata (Burm.f.) Wall. ex Nees is a medicinal plant of the family Acanthaceae and is popularly referred to as Kalmegh or “king of bitters”. It is widely cultivated and spread throughout tropical and sub-tropical countries, especially South and Southeast Asia. Traditionally, the plant has been used for inflammation, digestive tract ailments, liver disorders and fever. It has been used ethnomedicinally for long and is also being subjected to greater pharmacological studies, making it a potential candidate for natural-products-based drug discovery [11]. The therapeutic potential of A. paniculata is mostly linked to its diterpenoid lactones, notably andrographolide, being one of its key pharmacologically active ingredients. Andrographolide is a labdane-type diterpenoid lactone, which has been shown to have antioxidant, anti-inflammatory, immunomodulatory and other pharmacological properties in experimental systems. Other phytoconstituents like neo andrographolide, deoxy andrographolide and related diterpenoids could be responsible for the biological activity of the plant. Preclinical studies have been undertaken with Andrographolide-containing preparations in chemically induced hepatic injury, models of metabolic dysfunction, inflammatory hepatic injury, and hepatic fibrosis. So far, reported outcomes are the improvement of some biochemical markers, the reduction of oxidative damage, the modulation of inflammatory signaling and the attenuation of histological injury. The results, however, are still model-, preparation-, dose-, and time-specific. The molecular pharmacology of andrographolide is of special significance for the hepatoprotection since certain of its effects overlap with the primary mechanisms of liver disease progression. Experimental studies have suggested that the modulation of Nrf2-mediated antioxidant signaling, inhibition of NF-κB-mediated inflammatory signaling, regulation of pathways of cell survival, and disruption of profibrotic signaling pathways may all play a role [12]. All these actions indicate that andrographolide has activities that is not limited to direct radical scavenging but has an effect on several aspects of hepatic injury response. Still, some of these molecular targets must be validated in relation to each other, and it remains to be determined how much some of these targets are dependent on concentration and how each relates to human disease. However, Andrographolide has several translational challenges such as aqueous solubility, variable oral bioavailability, metabolic instability and lack of liver specific clinical efficacy data. Moreover, the safety profiles of various extracts of Andrographis, pure andrographolide and chemically modified derivatives are not necessarily the same. It is possible that selected pharmaceutical limitations can be overcome by advanced formulation technologies and structural modification strategies, but enhanced drug exposure does not necessarily lead to better hepatoprotective effects. In general A. paniculata can be considered as a good source of hepatoprotective bioactive molecules for hepatoprotective drug development. The, high pharmacological versatility makes it important to study Andrographolide in diseases with similar oxidative, inflammatory, metabolic and fibrotic processes. Its designation as a next generation hepatoprotective agent is however to be interpreted in a research context and not as a clinical designation. Molecular target validation, pharmacokinetic optimization, standardization of the preclinical models and well-designed human trials are required to determine the potential of andrographolide for a safe and effective therapy for liver diseases [13].

2. Botanical Profile and Phytochemistry of Andrographis paniculata

2.1. Taxonomy, Geographical Distribution, and Traditional Medicinal Uses

Andrographis paniculata (Burm.f.) Wall. ex Nees, also known as Kalmegh, green chiretta and the “king of bitters”, belongs to the family Acanthaceae (Lamiales). An annual erect highly branched herb with slender green stems, narrow, lanceolate leaves and minute white flowers with purple spots. Typically, it grows in tropical and subtropical climates and is known for its very bitter taste, notably attributed to diterpenoid lactones. The Royal Botanic Gardens, Kew states that it is native to the Indian subcontinent (India, Bangladesh, Nepal and Sri Lanka) and has been introduced in several areas of Southeast Asia and beyond. Traditional Chinese medicine, other ethnomedicine from Asia and traditional medicine use A. paniculata to treat fever, gastrointestinal disorders, respiratory infections, inflammation and liver disorders. According to Ayurveda, Kalmegh is used for bitter tonic preparations and for digestive and liver-related ailments such as jaundice. The aerial parts, including leaves, are commonly used for extracting powders, extracts and herbal medicines. Traditional uses also include the plant in formulations that nourish the appetite, digestion and functions of the body. These ethnomedicinal uses must be differentiated from the clinically proven therapeutic uses, however. Its geographical origin, environmental conditions, developmental stage, harvesting time, and procedure of extraction plays a significant role in the medicinal properties of A. paniculata. All these factors affect the concentration of andrographolide compounds and other metabolites, which may affect the biological activity and reproducibility of the herbal preparations. Therefore, botanical authentication, proper cultivation and phytochemical standardization are crucial factors to consider when developing consistent pharmaceutical products [14].

2.2. Major Phytochemical Constituents

A. paniculata contains a variety of secondary metabolites such as diterpenoid lactones, diterpenoid glycosides, flavonoids, phenolic compounds, xanthones and minor compounds. Ent-labdane diterpenoids are the most well-studied constituents of these chemical classes and are present in high concentrations and have a wide range of experimental pharmacological activities. Leaves, stems, roots and other aerial parts of the tree contain a variety of diterpenoids and flavonoids, which have been identified in phytochemical studies. They are present in varying proportions depending on the plant part, geographical origin, extraction solvent and post harvest processing [15]. Andrographolide is the main marker compound and one of the main biologically active compounds of A. paniculata. Other important diterpenoids are neoandrographolide, 14-deoxyandrographolide, 14-deoxy-11,12-didehydroandrographolide, andrograpanin and andrographoside. These compounds have differences in their hydroxylation, glycosylation, unsaturation, and other structural features that can affect their solubility, metabolic fate and interactions with biological targets. The sugar residues add on to the structural diversity of diterpenoid glycosides, which can alter (a) the physicochemical properties and (b) the pharmacokinetics. Another category of the secondary metabolites is flavonoids. Examples found are 7-O-methylwogonin, 5-hydroxy-7,2',3'-trimethoxyflavone and methoxylated flavone derivatives. Flavonoids have been of interest due to their common anti-oxidant and anti-inflammatory activity in experimental systems. However, the role of individual flavonoids in the hepatoprotective activity of A. paniculata is not well studied as compared to andrographolide. These constituents can be identified, quantified, and structurally characterized using modern analytical techniques such as HPLC, HPLC–MS, and NMR. These methods are especially significant for the identification of the chemical fingerprints and the setting of the quality-control standards for extracts to be used in pharmacological research or in pharmaceutical development [16].

2.3. Andrographolide: Chemical Structure and Physicochemical Properties

The major bioactive compound of A. paniculata is naturally occurring ent labdane diterpenoid lactone called andrographolide. It has a molecular formula of C??H??O? and a molecular weight of ~350.45 g/mol. Andrographolide has a bicyclic diterpenoid framework, includes an α,β-unsaturated γ-lactone ring and 3 hydroxyl groups. These features are involved in its chemical reactivity and interactions with biological macromolecules. The electrophilic unsaturated lactone system, in particular, has been studied for possible interaction with nucleophilic residues of proteins, but a direct experimental proof of a specific target interaction is still needed. Andrographolide is an ent-labdane diterpenoid lactone that is the main compound found in Andrographis paniculata. The andrographolide is usually present in solid form, in the form of a colorless or white crystalline compound, and is poorly soluble in water, an important problem for drug delivery by oral route [17]. The relatively lipophilic diterpenoid structure, together with several hydroxyl groups, may create a physicochemical profile that makes it difficult to dissolve, to transport through membranes and to formulate. Andrographolide has been shown to have a number of pharmacological properties in experimental models, but its aqueous solubility and systemic exposure are variable and might limit its therapeutic application. Anti-oxidant, Anti-Inflammatory, Immunomodulatory, Cytoprotective properties of the compound have been investigated. It is proposed to mediate the antioxidant defense through Nrf2 and to inhibit certain inflammatory signaling pathways. These properties suggest that andrographolide could be a potential scientific basis for studying it in various oxidative and inflammatory liver diseases. But, the molecular activity seen in experiments does not correlate with target engagement or therapeutic efficacy in the clinic. In order to overcome its biopharmaceutical drawbacks, structural derivatives, prodrugs and new delivery systems like polymeric nanoparticles, lipid based carriers and phospholipid complexes have been explored. The purpose of these strategies is to increase dissolution, stability, absorption or tissue distribution. Extensive pharmacokinetic and toxicological assessments are required to establish if such changes offer useful therapeutic benefits [18].

2.4. Other Bioactive Diterpenoids and Flavonoids

Andrographolide is the most well-studied compound in A. paniculata, but a number of other diterpenoids and flavonoids could be responsible for the plant's broader therapeutic profile. Neo andrographolide is a diterpenoid glycoside, which has been studied for its anti-inflammatory and antioxidant properties. It is glycosylated (not same as andrographolide) and it might affect its solubility, absorption and metabolic activity. However, there is still a lack of direct evidence of its independent hepatoprotective efficacy in clinical setting. 14-deoxyandrographolide (14-DA) and 14-deoxy-11,12-didehydroandrographolide (11,12-DDA) are structurally similar metabolites of A. donax which have been investigated for their biological activity in experimental systems. Molecular recognition and pharmacological activity can be altered by structural differences in the hydroxyl or double bond arrangement. Other minor diterpenoids, primarily andrograpanin, extend the chemical diversity of the plant, but have fewer and less well-defined liver-specific therapeutic properties. Another potentially relevant phytochemical fraction is flavonoids, including 7-O-methylwogonin and methoxylated flavone derivatives. They have both phenolic and oxygenated structures which could be involved in redox-related biological activity and modulation of inflammatory responses [19]. However, the finding of antioxidant activity in vitro does not necessarily predict any in vivo hepatoprotection. The pharmacological role of these compounds is dependent on absorption, metabolism, reach of tissue levels and the interaction with the cellular target. But importantly, the therapeutic activity of crude A. paniculata extracts cannot be solely credited to andrographolide until a proper design of constituent-depletion, fractionation and comparative pharmacological experiments are carried out. These interactions can be additive, synergistic or antagonistic, but these relationships need to be quantitatively validated. Advanced phytochemical profiling and comparative bioactivity assays in future studies, therefore, should be used to identify the most directly responsible constituents for hepatic protection. The methodology used here can help in standardizing and characterizing botanical preparations, as well as assist in the discovery of new hepatoprotective candidates based on andrographolide [20].

Table 1: Major Phytochemical Constituents of Andrographis paniculata: Chemical Classification, Molecular Targets, and Potential Hepatoprotective Activities

3. Pathophysiological Basis of Liver Disorders

3.1. Oxidative Stress and Mitochondrial Dysfunction

Oxidative stress is a significant mechanism of pathogenesis for liver diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease, drug-induced hepatotoxicity, and liver fibrosis. It is generated when the generation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is greater than the capacity of endogenous antioxidant defense systems. The liver is a special target for oxidative damage since it plays a key role in metabolizing exogenous chemicals (xenobiotics), in oxidizing lipids, and in producing energy through mitochondria. Overproduction of ROS comes from several intracellular sources such as the mitochondrial electron transport chain, cytochrome P450 enzymes, peroxisomes and NADPH oxidases. A set of antioxidant enzymes, including superoxide dismutase (SOD), catalase and glutathione peroxidase, control redox homeostasis under physiological conditions. But chronic exposure to metabolic stress, alcohol or hepatotoxic chemicals or inflammatory mediators can lead to derangements of these protective mechanisms contributing to lipid peroxidation, protein oxidation, and DNA damage. Mitochondrial dysfunction further deteriorates the oxidative injury by influencing the oxidative phosphorylation, decreasing the ATP production and disrupting the mitochondrial membrane potential. Oxidative damage can cause increased permeability of the mitochondrial membrane, release of cytochrome c and activation of intrinsic apoptosis pathways. Moreover, there is a link between impaired mitochondrial β-oxidation and intracellular lipid accumulation/lipotoxicity. Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is crucial to antioxidant defense of cells by controlling expression of genes coding for heme oxygenase-1 (HO-1), NAD (P)H quinone oxidoreductase 1 (NQO1) and other cytoprotective proteins. If such adaptive responses are continually surpassed by persistent oxidative stress, it can result in hepatocyte injury and inflammatory activation. Hence, maintaining integrity of mitochondria and balancing their redox state are promising strategies for decreasing liver injury and delaying the disease progression [21].

3.2. Chronic Inflammation and Immune Dysregulation

Chronic inflammation is one of the most important factors involved in the development of liver fibrosis, cirrhosis and HCC. A specialised immune environment exists within the liver, consisting of resident Kupffer cells, recruited monocytes, macrophages, dendritic cells, natural killer cells and adaptive immune populations. In normal physiology they have immune surveillance roles and promote tissue homeostasis. Excessive immune activation however, can be caused by exposure to lipotoxic metabolites, microbial products, viral infections, alcohol derived compounds, or damaged hepatocytes. When injured hepatocytes release damage-associated molecular patterns (DAMPs) to trigger inflammatory signaling pathways through pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4 and downstream nuclear factor-κB (NF-κB) signaling increases the transcription of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). These mediators attract immune cells, cause hepatocyte damage, and enhance local inflammation. Furthermore, activation of the NLRP3 inflammasome plays a role in the activation and maturation of caspase-1, as well as the release of IL-1β and IL-18. Persistent inflammatory signaling is able to disrupt the equilibrium of repair and regeneration, promoting a progressive remodeling of the liver. In addition to immune dysregulation, there is an altered phenotype of macrophages, excessive production of chemokines, and communication between innate and adaptive immune cells. While inflammatory responses play a protective role in pathogen clearance and tissue repair, chronic activation of inflammation can further damage hepatocyte and stimulate hepatic stellate cells. Thus, modulation of inflammatory cytokines and regulation of inflammasome activation are key targets in hepatoprotection drug development, along with NF-κB inhibition [22].

3.3. Hepatocyte Apoptosis, Necrosis, and Pyroptosis

Liver cell death is a hallmark pathological process of a number of acute and chronic liver diseases. Apoptosis, necrosis, pyroptosis, necroptosis and ferroptosis are distinct forms of cell death that have overlapping but different molecular mechanisms of liver damage. Apoptosis is regulated programmed cell death associated with cell shrinkage, chromatin condensation and activation of caspases. It can happen by the intrinsic mitochondrial pathway (Bax, Bcl-2, cytochrome c, and caspase-9) or by the extrinsic pathway of signalling via death-receptor binding molecules including Fas and TNF receptor 1. Increased apoptosis leads to a loss of functional hepatocyte numbers, and could contribute to inflammatory/fibrogenic responses through interactions with resident immune cells. In necrosis, on the other hand, the cell suffers serious injury, loss of cell membrane, swelling of the cell, and leakage of cell contents into the surrounding tissues. This leads to the release of DAMPs, which can further promote inflammation and activate further immune cells [23]. Necrotic hepatocyte injury is especially important in severe liver toxicity, ischemia and liver inflammatory diseases. Pyroptosis is gasdermin-dependent membrane pore opening regulated cell death that is inflammatory. Canonical inflammasome signalling is characterized by activation of NLRP3 leading to cleavage of gasdermin D (GSDMD) and subsequent release of IL-1β and IL-18. Pathways associated with pyroptosis have been linked to the following experimental models of liver injury: metabolic, alcohol-associated, or inflammatory. The degree of pyroptosis in individual cell types within the liver, however, is dependent on the disease etiology and experimental conditions. All of these cell death mechanisms contribute to oxidative stress and immune activation, establishing a vicious cycle of hepatocyte death, inflammation, and fibrosis. Therefore, therapeutic interventions that modulate mitochondrial apoptosis, inflammatory cell death, and cellular stress responses may provide additional hepatoprotective possibilities [24].

3.4. Lipid Accumulation and Metabolic Dysfunction

Excessive accumulation of lipids in the liver is a hallmark of MASLD, with a significant role in causing metabolic liver damage. In normal circumstances, the hepatic regulation of lipid homeostasis is accomplished by the balanced regulation of fatty acid uptake, de novo lipogenesis, mitochondrial β-oxidation and secretion of VLDL. However, an insulin resistance, excess calorie consumption, adipose tissue dysfunction, and lipid metabolism imbalance can disrupt these processes, leading to an intracellular accumulation of triglycerides. A greater flux of fatty acids from adipose tissue and greater hepatic lipogenesis are important factors driving fat accumulation, and a reduction in lipid oxidation and export also play a role in lipid retention [25]. Importantly, the primary cause of hepatocellular injury may not be hepatic triglyceride storage. Lipotoxicity, endoplasmic reticulum stress, mitochondrial dysfunction, and inflammatory signaling could be triggered by the excessive accumulation of potentially toxic lipid species, such as certain saturated fatty acids, ceramides, and free cholesterol. The regulation of hepatic lipid metabolism involves Sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and peroxisome proliferator-activated receptors (PPARs). AMP-activated protein kinase (AMPK) is a cellular energy sensor that is capable of inhibiting fatty acid synthesis and stimulating fatty acid oxidation via downstream molecular pathways. Chronic metabolic disease leads to the development of relatively benign steatosis into steatohepatitis, which is associated with hepatocellular injury, inflammation and some degree of fibrosis. This is further exacerbated by altered insulin signaling and chronic low grade inflammation. Thus, the restoration of hepatic metabolic homeostasis through AMPK activity regulation, lipid biosynthesis, mitochondrial fatty acid oxidation and insulin sensitivity are of significant therapeutic interest in metabolic liver diseases [26].

3.5. Hepatic Stellate Cell Activation and Fibrogenesis

Hepatic fibrosis is a maladaptive wound-healing reaction to continual hepatocellular damage and chronic inflammation. In the liver, the process is regulated by hepatic stellate cells (HSCs), which are mainly located in the space of Disse. During normal conditions, the HSCs are largely quiescent, and play a role in storage of vitamin A and in the homeostasis of the extracellular matrix. After repeated hepatic insults, they are activated and differentiated to become cells that are proliferative and have a myofibroblast-like shape, due to the action of inflammatory cytokines, ROS, apoptotic signals and profibrotic mediators. The activated HSC is expressing α-smooth muscle actin (α-SMA) and secreting excessive amounts of extracellular matrix (ECM) proteins, especially type I and type III collagen. Transforming growth factor-β1 (TGF-β1) is a primary profibrotic factor, which activates Smad2/3 dependent transcriptional activity, and stimulates collagen production. Another factor, platelet-derived growth factor (PDGF) stimulates proliferation, migration and survival of HSCs. Other pathways also regulate fibrogenic responses such as MAPK, PI3K/Akt and oxidative stress pathways [27]. The deposition of the extracellular matrix causes changes in liver architecture, higher intrahepatic vascular resistance, and ultimately leads to portal hypertension and cirrhosis. Fibrosis also involves an imbalance between the production and degradation of extracellular matrix. Regulation of MMP/tissue inhibitors of MMP (TIMP) promotes collagen deposition and defective tissue remodeling. Recovery from hepatic fibrosis can be achieved if the underlying cause is treated effectively, but when fibrosis is advanced recovery can be limited by underlying architectural damage and ongoing profibrotic signals. Therefore, drugs that inhibit activation of pathological HSC, block TGF-β1/Smad signaling, and repair ECM homeostasis are potential therapeutic options for stopping the progression of hepatic fibrosis [28].

3.6. Gut–Liver Axis Dysregulation

The gut–liver axis is a complex bidirectional communication system between intestinal microbiota, the intestinal epithelial barrier, portal circulation, immune signaling and hepatic metabolism. The liver is exposed to nutrients, microbial metabolites and microbial associated molecular patterns continuously, as it receives a significant amount of blood from the gastrointestinal tract via the portal vein. In healthy gut, the state of intestinal barrier integrity and a balanced microbial community is linked to liver immune tolerance and metabolic health. But, disturbance of this balance can occur when there is intestinal dysbiosis, increased intestinal permeability, and changes in the metabolism of the micro-organisms, all of which can add to hepatic inflammation [29]. Dysbiosis can alter the production of metabolites by the microbes, such as short chain fatty acids, secondary bile acids, ethanol, and other metabolites that affect hepatic lipid metabolism, inflammatory signaling and metabolic regulation. Defective intestinal barrier function may lead to the translocation of intestinal bacterial components, such as LPS to portal circulation. LPS-induced activation of TLR4 signaling in Kupffer cells and other hepatic cell subsets leads to the generation of inflammatory cytokines, and may be involved in fibrogenic responses. The mechanisms of bile acid changes in its composition and signaling through farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) might also modify lipid, glucose, and immune functions. The gut–liver axis is therefore involved in the pathogenesis of MASLD, ALD and some forms of advanced chronic liver injury. Alterations in the microbiome, however, are very different between diseases and individuals and therefore universal therapeutic targets are challenging. A range of new strategies are being explored to restore gut–liver communication, including dietary manipulation, microbiota-directed interventions, intestinal barrier protection and modulation of bile acid signaling. These mechanisms can serve as a general background for the identification of other potential hepatoprotective targets. These are all interdependent pathological processes and together they show that molecular abnormalities are not sufficient to explain liver disorders. All of these contribute to disease progression in a complex way, known as oxidative stress, mitochondrial injury, inflammatory activation, dysregulated hepatocyte death, metabolic disturbances, fibrosis, and gut-derived signals. The overlapping of their signaling networks gives a mechanism to consider multi-target compounds such as andrographolide. However, therapeutic relevance of such compounds will need to be directly validated with targets, good liver exposure, and efficacy in proper disease models before therapeutic benefit can be established [30].

4. Molecular Mechanisms Underlying Andrographolide-Mediated Hepatoprotection

4.1. Regulation of Oxidative Stress Through the Nrf2/Keap1/HO-1 Pathway

Nrf2/Keap1/HO-1 pathway is an important pathway regulating oxidative stress. Oxidative stress is a significant factor in hepatocellular injury, especially in metabolic dysfunction-associated steatotic liver disease (MASLD), drug-induced liver injury and hepatic fibrosis. A bioactive diterpenoid lactone present in Andrographis paniculata (andrographolide) has shown antioxidant and cytoprotective effects by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. In the normal state, Nrf2 is regulated by Kelch-like ECH-associated protein 1 (Keap1), which helps to promote its ubiquitination and proteasomal degradation. Under oxidative stress, the failure to regulate Nrf2 by Keap1 allows Nrf2 to accumulate and translocate into the nucleus to activate antioxidant response element associated genes [31]. These include heme oxygenase-1 (HO-1), NAD (P)H quinone oxidoreductase 1 (NQO1) and enzymes for glutathione synthesis and regeneration. It has been reported that andrographolide can improve the antioxidant responses under Nrf2 regulation, which leads to the decreased accumulation of reactive oxygen species (ROS) and lipid peroxidation in experimental models of liver injury. In acute liver injury, induced by lipopolysaccharide/D-galactosamine, andrographolide restored the levels of Nrf2 and HO-1, and lowered malondialdehyde levels, inflammatory cytokines and serum transaminase activity. Other hepatocyte studies have suggested that the p38 MAPK/Adenosine A2AR pathway and inhibition of glycogen synthase kinase-3β are involved in maintaining nuclear activity of Nrf2. The results indicate that andrographolide can activate the host antioxidant mechanism instead of being just a direct free-radical scavenger. Direct modification of Keap1 by andrographolide and the relative contribution of each upstream mechanism, however, needs to be confirmed in models of disease [32].

4.2. Suppression of Inflammatory signaling Through TLR4/NF-κB

Chronic inflammatory signaling is tightly linked to the progressive hepatocellular damages and fibrogenesis. The toll-like receptor 4 (TLR4) detects lipopolysaccharide (LPS) and other inflammatory stimuli, promoting recruitment of adaptor molecules, activation of the downstream kinases and stimulation of nuclear factor-kB (NF-kB). In inflammatory conditions, the degradation of inhibitory proteins, IκB, exposes the NF-κB subunits that then enter the nucleus to regulate gene transcription of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase and other immune-response molecules. This signaling network has been shown to be targeted by andrographolide, which has anti-inflammatory effects. With carbon tetrachloride (CCl4) liver fibrosis, liver fibrosis was found to be decreased, along with inflammatory markers, serum aminotransferases and NF-κB p50 signaling, which was associated with reduced hepatic expression of TLR4. This is similar to the suppression of NF-κB activation seen in experimental acute liver injury [33]. This indicates that andrographolide can act as an inhibitor of inflammatory amplification, which involves recruited immune cells, resident macrophages, and activated hepatic stellate cells. The reduced production of inflammatory mediators may then help to decrease the oxidative damage and profibrotic stimulation. However, the exact upstream interactions involved in the TLR4/NF-κB inhibition are still not fully understood and, therefore, the experimental anti-inflammatory activity is not necessarily a reflection of clinical activity during the chronic phase of liver disease.

4.3. Modulation of MAPK Signaling Pathways

Signaling pathways that include mitogen-activated protein kinases (MAPK) control proliferation, differentiation, inflammatory processes, apoptosis and response to environmental stress in cells. The main MAPK families are extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK. The functions of these kinases include modulation of inflammatory receptors, oxidative responses, mitochondrial signaling, and activation of hepatic stellate cells, all of which lead to liver damage. In contrast, sustained activation of JNK may enhance lipotoxicity and apoptotic signals, while p38 and ERK pathways have varying and context-dependent effects on cellular survival and inflammation [34]. The use of andrographolide as a modulator of MAPK-associated processes has been explored, but the outcome of these studies depends on cell type, disease model, concentration and length of treatment. Importantly, MAPK inhibition cannot be considered as its universal mechanism. HepG2 cells exposed to hydrogen peroxide were used to explore the protective property of the andrographolide-induced activation of p38 MAPK, manifested as the nuclear translocation of Nrf2 and its increased expression. However, a decrease in excessive MAPK activation due to suppression could be helpful in other inflammatory conditions. This apparent complexity emphasizes the need to consider the phosphorylation status of individual kinases, and not to simplify the signalling of MAPKs as either "on" or "off. To determine if modulation of ERK, JNK or p38 is directly responsible for hepatoprotection resulting from andrographolide treatment, additional investigations in the liver are needed that employ pharmacological inhibitors, gene silencing, and time-dependent phosphorylation studies [35].

4.4. Regulation of NLRP3 Inflammasome Activation and Pyroptosis

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is an important regulator of innate immune activation and inflammatory cell death. After proper priming and activation signals, NLRP3 assembles to a multiprotein complex with apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspase-1. Activated caspase-1 then triggers the maturation of IL-1β and IL-18 and the cleavage of gasdermin D (GSDMD). This causes gasdermin-induced pore formation in the cell membranes which leads to the release of inflammatory mediators and pyroptosis of cells, further enhancing liver inflammation. Experimental studies suggest that andrographolide is able to reduce liver damage by blocking this pathway [36]. In 2025, researchers explored the issue of acute liver damage due to lipopolysaccharide (LPS) in rats and BRL-3A hepatocyte cells stimulated with ATP and LPS. Liver injury indices were improved and the expression of mediators of NLRP3/caspase-1/GSDMD signaling were decreased following andrographolide treatment. Additionally, network pharmacology and molecular docking revealed potential interactions with proteins associated with the inflammasome. However, the computational binding predictions are not proof of biochemical target engagement. The mechanistic link between these findings and further work with genetic knockout models, quantitative assessment of pyroptotic membrane permeabilization, and target-rescue experiments is still required to confirm pathway dependence and the relevance of this mechanism in chronic hepatic diseases.

4.5. Modulation of Apoptosis and Mitochondrial Homeostasis

Modulation of Apoptosis and Mitochondrial Homeostasis is an approach that targets the mechanisms of cell death and energy production in mitochondria. Under inflammatory, metabolic and toxic conditions, mitochondrial dysfunction and increased hepatocyte apoptosis play a significant role in liver injury. Antiapoptotic Bcl-2 and proapoptotic Bax and Bak proteins of the B-cell lymphoma 2 (Bcl-2) family regulate the intrinsic pathway of apoptosis. The outer mitochondrial permeabilization is the result of severe mitochondrial damage, leading to the release of cytochrome c, formation of apoptosome, and activation of executioner caspases [37]. Mitochondrial dysfunction in terms of respiration, ATP production and calcium regulation may exacerbate hepatocellular injury. In certain experimental models of nonmalignant liver damage, andrographolide has been shown to have antiapoptotic properties. For instance, andrographolide inhibited the apoptosis and oxidative stress of hepatocytes in immune-mediated liver damage caused by concanavalin A (Con A) treatment. This protection could occur, in part, because of the reduction in oxidative damage and inflammatory signaling, which lessens the stimuli leading to the damage of mitochondria. However, there are only indirect arguments supporting this view of mitochondrial preservation as the main mechanism. Critically, andrographolide could have opposite actions in malignant liver cells, where induction of apoptosis is potentially beneficial. Unlike other context-dependent activities, this activity highlights the difference between protecting healthy hepatocytes and suppressing hepatocellular carcinoma cells. The effect of it on Bax/Bcl-2 ratio, activation of caspases, mitochondrial membrane potential and mitochondrial quality-control mechanisms needs to be considered separately in both nonmalignant and malignant liver models [38].

4.6. Regulation of AMPK/mTOR Signaling and Autophagy

Autophagy is a highly conserved process that occurs within cells to eliminate excess cell components, mis-folded proteins and damaged organelles. This is of special significance in hepatic metabolism due to the ability to regulate lipid levels, mitochondrial function, oxidative stress, and inflammatory damage because of impaired autophagic activity. AMP-activated protein kinase (AMPK) is an energy-sensing regulator which is activated when the level of the nucleotide in cells changes. In the context of energy stress, AMPK may enhance adaptive responses and metabolic homeostasis by stimulating the activation of a class of autophagy-activating kinases autophagy activating kinase 1. In an energy stress situation, AMPK can stimulate the activation of a class of autophagy-activating kinases autophagy activating kinase 1 to promote adaptive responses and metabolic homeostasis. In a few experimental systems, andrographolide has been studied for its ability to impact energy metabolism and autophagic pathway related signaling. The results suggest a rationale for investigating the hepatoprotective effect of AMPK/mTOR modulation. But its direct effects on autophagic flux in particular diseases in the liver are not as well established as its anti-oxidant and anti-inflammatory effects. Additionally, cell population determines different effects of autophagy. The appropriate removal of damaged mitochondria and excess lipid droplets by an adequate autophagic activity is beneficial in hepatocytes; in contrast, autophagic-dependent lipid mobilization in hepatic stellate cells could be beneficial to their activation and fibrogenic transformation. Therefore, in all pathological situations, the stimulation of autophagy can be dangerous. The turnover of LC3-II, the degradation of p62/SQSTM1, lysosomal function and autophagic flux should be measured in future studies in order to establish causal relationships using AMPK- and mTOR-specific interventions [39].

4.7. Inhibition of Hepatic Fibrosis Through TGF-β1/Smad Signaling

Hepatic fibrosis represents an abnormal accumulation of extracellular matrix in the liver's tissues that occurs after prolonged liver damage. One of the major events in this process is the activation of hepatic stellate cells (HSCs) to collagen-producing myofibroblast-like cells. Transforming growth factor-β1 (TGF-β1) induces fibrogenesis by activation of its receptors and phosphorylation of Smad2 and Smad3 which are responsible for the regulation of the transcription of collagen-associated genes and other profibrotic mediators. Persistent signaling leads to elevated expression of α-smooth muscle actin (α-SMA), hepatic extracellular matrix accumulation and progressive hepatic architectural distortion [40]. In experimental models where this pathway is engaged, andrographolide has been shown to have antifibrotic effects. In a carbon tetrachloride-induced mouse model, andrographolide inhibited collagen deposition, serum aminotransferase activities, α-SMA expression, and activation of the TGF-β1 pathway. Additional experiments with LX-2 and primary HSCs also showed a reduction in Smad2 phosphorylation, though Smad3 phosphorylation did not appear to be significantly affected under the conditions reported. This difference suggests that the compound can have pathway- and model-specific effects, rather than inhibit all Smad proteins. A 2024 experimental study showed that the use of andrographolide plus pirfenidone led to greater antifibrotic activity than either agent alone in the biliary fibrosis model studied. Together with these effects, the activation of HSCs and TGF-β/Smad signaling were decreased. While these are encouraging, further studies are needed to confirm the results and to investigate potential interactions between the drugs before any therapeutic combinations can be recommended on a clinical basis [41].

4.8. Regulation of Hepatic Lipid Metabolism Through AMPK/SREBP-1c/PPAR Pathways

The hepatic lipid metabolic dysfunction is a hallmark of metabolic dysfunction-associated steatotic liver disease. These factors all contribute to hepatic triglyceride accumulation: excessive fatty acid uptake, and increased de novo lipogenesis, impaired mitochondrial β-oxidation, and insulin resistance. These are regulated by several regulatory proteins, such as AMPK, sterol regulatory element-binding protein-1c (SREBP-1c), and the peroxisome proliferator-activated receptors (PPARs). Oxidation of fatty acids also activates AMPK, which is a general inhibitor of energy-consuming lipid synthesis, while the transcription of lipogenic enzymes like fatty acid synthase and acetyl-CoA carboxylase is promoted by the activation of SREBP-1c. PPARα promotes fatty acid oxidation, and PPARγ has tissue- and context-specific effects on insulin sensitivity and lipid storage. Andrographolide has been shown to have antisteatotic effects in experimental metabolic liver disease. In high fat diet-fed mice, it decreased the hepatic steatosis and enhanced multiple metabolic parameters. Mechanistic studies revealed that suppression of fatty acid uptake via fatty acid transport protein 2 (FATP2) to be an important aspect of this response. Andrographolide also decreased the expression of SREBP-1c and lipogenic enzymes in the hepatic cells treated with oleic acid. Importantly, its lipid-lowering effect was reduced by FATP2 overexpression, giving more convincing mechanistic evidence than just an association with changes in the expression of the genes. The AMPK and PPAR pathways are potential additional mechanisms for protection, but the contribution of each pathway to the protection by andrographolide needs to be directly verified [42].

4.9. Modulation of PI3K/Akt Signaling

The phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway is involved in cellular survival, glucose metabolism, proliferation, inflammatory response and apoptosis. PI3K activation stimulates the synthesis of phosphatidylinositol-3,4,5-trisphosphate which allows the recruitment and phosphorylation of Akt. Activated Akt then regulates multiple downstream targets such as glycogen synthase kinase-3β, forkhead box O transcription factors and mTOR signal transduction. This pathway plays a critical role in hepatic tissues, where it relates to insulin sensitivity, metabolic regulation, hepatocyte survival, and tissue regeneration. Because of its interaction with oxidative stress, autophagy and inflammatory pathways, possible involvement of PI3K/Akt signaling in andrographolide-associated hepatoprotection has garnered attention. Based on network pharmacology analysis, AKT1 was identified as a potential molecular target of andrographolide in ALI. However, this does not provide computational target identification, functional regulation or direct binding. Moreover, the activation of Akt can promote the recovery of injured nonmalignant hepatocytes, and can promote the survival and proliferation of malignant cells in the liver cancer, namely hepatocellular carcinoma. Therefore, the therapeutic potential of PI3K/Akt modulation will depend on the stage of the disease, the cellular context and the downstream signaling events. More studies using selective Akt inhibitors, genetic target validation and Akt phosphorylation analysis are needed to confirm its role in the liver effects of andrographolide [43].

4.10. Emerging Roles of MicroRNAs and Epigenetic Regulation

There is an increasing importance of role of microRNAs (miRNAs) and epigenetic mechanisms in regulation of hepatic inflammation, metabolism, fibrosis and carcinogenesis. MicroRNAs are short (non-coding) RNA molecules that control gene expression by post-transcriptional regulation of target messenger RNAs. Alteration in hepatic miRNAs also plays a role in various liver disorders, such as lipid metabolism, oxidative stress, activation of HSC, apoptosis etc. Cellular responses to chronic metabolic and inflammatory stresses are also modulated by epigenetic mechanisms such as DNA methylation, histone modifications, and chromatin remodeling. Recent studies indicate that andrographolide can modulate some regulatory networks of RNA that are important for hepatic cytoprotection. Experimental data (summarized in mechanistic reviews) suggest andrographolide has a regulatory effect on the hepatocyte nuclear factor 4α associated regulation of miR-433 and miR-377, which are associated with antioxidant response, including glutathione (GS) and HO-1. Further research works have been mentioned about possible associations between andrographolide, regulation of miRNA, hepatic fibrosis and liver cancer. However, all the evidence is still preliminary and specific miRNA targets for each disease have not been validated thoroughly. While epigenetic modulation is another potential mechanism of action, there is still not enough direct evidence to show that andrographolide-induced epigenetic changes in the hepatic DNA or histone-modifying enzymes are the general mechanism by which it promotes hepatoprotection. The combined analysis of transcriptomics, small-RNA sequencing, chromatin accessibility and functional gene-silencing analyses in the future will provide new insights into regulatory networks and how they are relevant to therapy. These methods can also help identify molecular markers that predict treatment outcomes and enable more targeted assessment of andrographolide therapies [44].

5. Therapeutic Potential of Andrographis paniculata in Liver Disorders

5.1. Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)

Metabolic dysfunction-associated steatotic liver disease (MASLD), a condition formerly classified as nonalcoholic fatty liver disease (NAFLD), is a liver condition associated with cardiometabolic abnormalities, such as obesity, insulin resistance, dyslipidemia, and type 2 diabetes mellitus, as well as excessive hepatic fat accumulation. Pathogenesis is through upregulation of hepatic uptake of fatty acids, augmented de novo lipogenesis, mitochondrial dysfunction, oxidative stress and dysregulation of inflammatory signaling. These closely related abnormalities facilitate the accumulation of lipid within the hepatocytes and can lead to inflammation and fibrosis. Therefore, bioactive molecules with the ability to modulate several metabolic and inflammatory pathways are gaining interest as possible therapeutic targets. The main diterpenoid lactone of Andrographis paniculata is andrographolide which has shown potential for antisteatotic activity in experimental models. A study feeding mice a high-fat diet for eight weeks demonstrated that andrographolide treatment at 50, 100 and 200 mg/kg/day improved glucose tolerance, insulin sensitivity and some metabolic parameters and reduced hepatic steatosis. Mechanistic studies showed that andrographolide inhibited the expression of fatty acid transport protein 2 (FATP2) resulting in the suppression of fatty acid uptake in the liver cells. Importantly, FATP2 overexpression reduced oleic acid-induced lipid-lowering effect in LO2 cells, supporting the notion that FATP2 regulation is linked to lipid-lowering effect in oleic acid-treated LO2 cells. Further experimental studies indicate that andrographolide has the potential to affect sterol regulatory element-binding proteins and enzymes involved in hepatic lipogenesis. The antioxidant and anti-inflammatory properties could also reduce the secondary damage caused by the lipid accumulation. However, the relative importance of the lipid transport regulation, modified lipogenesis and metabolic improvement needs to be further understood. However, while the results from the prior studies suggest therapeutic potential, the majority have used experimental models that have previously been termed NAFLD, instead of those defined by the modern criteria used for the diagnosis of MASLD. In addition, there are dosage differences between animals and humans for therapeutic use. As such, well-designed clinical studies are needed to establish the potential of andrographolide in achieving clinical benefits with respect to hepatic steatosis, metabolic markers, and long-term clinical outcomes [45].

5.2. Metabolic Dysfunction-Associated Steatohepatitis (MASH)

Metabolic dysfunction-associated steatohepatitis (MASH), also known as nonalcoholic steatohepatitis (NASH), is a form of progressive inflammatory liver disease that includes hepatic steatosis and ballooning, lobular inflammation, and variable fibrosis. The progression is linked to lipotoxicity, mitochondrial damage, generation of reactive oxygen species, recruitment of macrophages and activation of hepatic stellate cells. Chronic inflammation and fibrosis can lead to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Therapies that inhibit both inflammatory and fibrotic processes are of special interest. In experimental steatohepatitis, andrographolide has shown to have beneficial effects by modulating inflammatory and fibrogenic pathways. Andrographolide (1 mg/kg, 3 times/week) decreased hepatic triglyceride accumulation, inflammation and fibrosis in mice fed a choline deficient, amino acid defined diet. Treatment also reduced the infiltration of macrophages and levels of pro-inflammatory and profibrotic gene expression. Mechanistic observations suggested that inhibiting nuclear factor-κB (NF-κB) signaling and dampening inflammation activation was implicated. Andrographolide inhibited the expression of IL-1β in Lipopolysaccharide-treated Lipid-loaded HepG2 cells and blocked the inflammatory response associated with the activation of the inflammasome. The results suggest that andrographolide may impact multiple mechanisms that contribute to the progression of hepatic steatosis to inflammatory and fibrotic liver damage. Specifically, the inhibition of NF-κB-dependent cytokine production and the down-regulation of activation of the NLRP3 inflammasome could limit the inflammatory amplification, and indirect blockade of profibrotic signaling could prevent the deposition of the extracellular matrix. Nevertheless, dietary models that lack a choline-diet do not adequately mimic the obesity, insulin resistance and metabolic parameters seen in human MASH. Thus, it is crucial to validate in metabolically representative models. To conclude, further studies are needed to evaluate disease activity, fibrosis regression, pharmacokinetic exposures and sustained therapeutic responses before andrographolide becomes a clinically useful drug for treating MASH [46].

5.3. Alcohol-Associated Liver Disease

Alcohol-associated liver disease is a spectrum of disease from hepatic steatosis, alcohol-associated hepatitis, through to progressive fibrosis and cirrhosis. Chronic ethanol exposure alters hepatic lipid metabolism and leads to oxidative injury via alcohol dehydrogenase, aldehyde dehydrogenase and met ac-2E1 mediated pathways. Hepatocellular injury is caused by the combined effect of acetaldehyde accumulation, increased production of reactive oxygen species, mitochondrial dysfunction and impaired intestinal barrier. Furthermore, gut-derived LPS can stimulate inflammatory signaling via Toll-like receptors and NF-κB, and activate Kupffer cells. Experimental studies indicate that andrographolide has a beneficial effect in reducing the hepatic injury induced by ethanol consumption, possibly due to its anti-oxidative and anti-inflammatory properties. Oral administration of andrographolide in chronic ethanol-exposure mouse model was found to benefit hepatic biochemical markers and histopathological alterations. The lipid accumulation, production of ROS and lipid peroxidation were found to be reduced in the treated animals. These enhancements were accompanied by reduced expression of the NF-κB and tumor necrosis factor-α protein, indicating that hepatoprotection involves inhibition of inflammatory signaling. In the past, another study focused on androgenic agents extracted from A. paniculata, andrographolide and arabinogalactan proteins, in an acute ethanol-toxicity mouse model was performed. These both exhibited protective action against the selected biochemical abnormality related to ethanol exposure, which suggests that the biological activity of the plant can not be solely attributed to andrographolide. Experimental observations with isolated compounds or extracts however, should not be taken to represent equivalent clinical activity. The results together suggest that further studies of andrographolide as an adjunctive therapy agent to alcohol-associated hepatic injury are warranted. However, there is limited evidence for the use of A. paniculata as a current treatment for alcohol-associated hepatitis or cirrhosis. Alcohol abstinence, nutritional treatment and the proper medical treatment of the disease is still primary treatment [47].

5.4. Drug-Induced Liver Injury

Acute liver injury due to drugs (DILI) is a significant cause of acute hepatic dysfunction and can be caused by predictable dose dependent toxicity, idiosyncratic reactions from therapeutic agents or both. One of the most well-studied experimental models is acetaminophen-induced hepatotoxicity. High acetaminophen exposure leads to the production of N-acetyl-p-benzoquinone imine (NAPQI) that consumes glutathione, damages mitochondrial proteins, increases oxidative stress, and leads to hepatocellular necrosis. These events can develop to acute liver failure if the liver cannot keep up with the hepatocellular damage. In a few chemically induced injury models, the hepatoprotective potential of A. paniculata and its diterpenoids has been investigated. Preliminary studies showed that andrographolide and neoandrographolide were protective against certain experimental hepatotoxic stimuli. The potential use of andrographolide formulations in the treatment of drug-induced liver injury in acetaminophen was investigated in subsequent studies, with a focus on antioxidant restoration and drug delivery. Using poly (lactic-co-glycolic acid) nanoparticles functionalized with heparin, the researchers found that nanoparticles carrying andrographolide were more efficiently delivered to the liver and protected against acetaminophen-induced liver injury in mice. Antioxidant capacity and hepatic glutathione stores were seen to be restored in the observed effects. When tested alone, andrographolide nanocrystals showed better pharmaceutical properties and greater hepatoprotective effects at lower dose than the unformulated andrographolide in mice. The findings in these studies suggest that the activity of these formulations could also affect the pharmacological effect due to different exposure of the liver. Although promising results have been reported, andrographolide has not yet proven to be a clinical antidote for acetaminophen toxicity. N-acetylcysteine continues to be the DTR treatment; other supportive therapies as appropriate depending on clinical severity. Furthermore, DILI is a group of mechanistically different disorders and protection against acetaminophen toxicity does not apply to idiosyncratic or immune-mediated adverse drug reactions. Andrographolide, therefore, should be investigated for its disease-specific responses and therapeutic timing, as well as possible interactions with other drugs and safety, for potential use in clinical applications for DILI [48].

5.5. Viral Hepatitis and Inflammatory Liver Injury

Viral hepatitis still poses a significant risk as a cause of chronic liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma. Hepatitis B and C viruses can cause chronic infections with immune-mediated damage to hepatocytes, and a progressive process of hepatocyte tissue remodeling. There are interactions occurring between viral replication, host immune responses, inflammatory cytokine activity, oxidative stress and processes of regeneration which determine the extent of hepatic injury. While clinically useful antiviral treatments have greatly improved clinical outcomes, the search for new antiviral and inflammation-modulating agents remains an area of active research. Experimental antiviral activity has been found for several of the constituents of A. paniculata. In 2014, a structure–activity relationship study revealed that andrographolide and dehydroandrographolide are inhibitors of replication of hepatitis B virus (HBV). The experimental half-maximal inhibitory concentration of andrographolide was 54.07 μM, and some chemically modified derivatives showed higher antiviral selectivity. Based on these observations, it is concluded that the diterpenoid scaffold is potentially promising for further successful antiviral lead optimization, albeit activity at micromolar concentrations does not imply clinically attainable antiviral activity. In addition to its antiviral research, andrographolide is also protective in experimentally induced inflammatory liver injuries. Treatment decreased serum aminotransferase activity, oxidative stress markers and proinflammatory cytokines in a lipopolysaccharide/D-Galactosamine mouse model, increased Nrf2 and heme oxygenase-1 expression, and inhibited NF-κB activation. These results suggest that it may have the capacity to regulate inflammatory injury without any direct antiviral activity [49].

5.6. Hepatic Fibrosis and Cirrhosis

Hepatic fibrosis is a progressive wound healing reaction to chronic liver cell injury that is the result of excessive extracellular matrix deposition, particularly of fibrillar collagen. One of the main mechanisms of fibrogenesis is activation of hepatic stellate cells (HSC) to become myofibroblast-like cells (MFLC). These activated cells express α-smooth muscle actin (α-SMA) and synthesize collagen in response to transforming growth factor-β1 (TGF-β1), platelet-derived growth factor, inflammatory cytokines and oxidative stress. The progression of ECM accumulation results in disruption of hepatic architecture and can eventually lead to cirrhosis, portal hypertension and hepatic insufficiency. Andrographolide has been shown to have antifibrotic effects in a variety of experimental models. Andrographolide decreased serum alanine aminotransferase and aspartate aminotransferase activity, collagen deposition, α-SMA expression and TGF-β1 associated signaling in mice with carbon tetrachloride-induced fibrosis [50]. In fibrotic mice, andrographolide was found to decrease the activity of serum alanine aminotransferase, aspartate aminotransferase, collagen deposition, and α-SMA expression and TGF-β1 associated signaling. LX-2 and primary hepatic stellate cells were used for complementary experiments that showed the inhibition of both TGF-β1/Smad2 and TLR4/NF-κB p50 pathways. The present results indicate that andrographolide is able to regulate inflammatory responses and stellate cell activation simultaneously, which may result in attenuation of fibrogenesis. Further data showed decreases in collagen accumulation in liver, inflammatory mediators, neutrophil infiltration, and angiogenesis-related signaling using a mouse model of thioacetamide-induced liver fibrosis. It also suppressed the expression of hypoxia- and VEGF signalling-related markers, indicating that its antifibrotic activity may be related to an interplay between inflammation, hypoxia and abnormal vascular remodeling. One of the major mechanistic pieces of evidence is related to acetaminophen-induced hepatic fibrosis, where it was revealed that andrographolide increased Nrf2-mediated antioxidant signaling and decreased stellate cell activation. Importantly, the protective effect was less pronounced in Nrf2 deficient mice, suggesting that this antioxidant pathway is important for protection and not merely correlated to changes in the expression of this protein [51-53].

Future Perspectives

The antioxidant, anti-inflammatory, metabolic regulatory, and anti-fibrotic activities of Andrographis paniculata and its primary bioactive compound, andrographolide, have made them promising agents for the development of new hepatoprotective drugs. Although promising results from experimental studies have emerged, numerous pharmacological, mechanistic, and translational hurdles remain which need to be overcome before these compounds can be considered clinically validated therapeutic agents. Further studies should focus on detailed molecular studies to elucidate the exact molecular interaction of andrographolide with signaling pathways such as Nrf2/Keap1/HO-1, TLR4/NF-κB, NLRP3 inflammasome, AMPK/mTOR, and TGF-β1/Smad. Emerging techniques such as transcriptomics, proteomics/protein profiling, metabolomics, gene silencing and CRISPR-based functional studies can assist in identifying with certainty direct molecular targets and differentiating primary pharmacological effects from secondary cellular response. Another research stream is finding ways to surmount the pharmaceutical constraints of andrographolide, such as its poor aqueous solubility, variable oral bioavailability, and its extensive metabolic transformation. The use of innovative delivery platforms such as polymeric nanoparticles, phytosomes, liposomes, solid lipid nanoparticles, and stimuli-responsive nanocarriers, should be carefully evaluated for its stability, absorption, and liver-specific accumulation. Better formulation properties, however, should be accompanied by comparative pharmacokinetic studies, biodistribution analysis, and experiments showing the therapeutic effects. Additionally, the study of the effect of andrographolide on gut–liver axis regulation, mitochondrial quality control, ferroptosis, immunometabolic signaling and epigenetic modifications should be explored further. These evolving mechanisms might offer further clues to its possible therapeutic utility in metabolic dysfunction-associated steatotic liver disease, steatohepatitis, drug-induced liver injury and hepatic fibrosis. Moreover, comparative investigations of the purified andrographolide, chemically modified derivatives, and standardized A. paniculata extracts are crucial to ensure the relative efficacy, safety, and pharmacological uniformity of the extracts. Perhaps most importantly, clinical translation will need to represent well-designed, randomized controlled trials utilizing standardized formulations, dosing, pharmacokinetics, validated liver markers, and clinically relevant endpoints. Special consideration should be given to long-term toxicity, herb–drug interactions, and the safety of patients with impaired hepatic function. Combining computational drug discovery, innovative formulation approaches, mechanistic studies, and evidence-based clinical evaluation could ultimately determine if andrographolide emerges as a viable hepatoprotective drug derived from natural sources.

CONCLUSION

In particular, the presence of a wide range of pharmacological activities in the principal diterpenoid lactone of andrographis, andrographolide, has made it a subject of great scientific interest as a source of new hepatoprotective substances. From the available preclinical evidence, andrographolide seems to exert multiple molecular mechanisms that contribute to the attenuation of hepatic injury, such as the inhibition of oxidative stress, modulation of inflammatory responses, preservation of mitochondrial function, modulation of lipid metabolism, and inhibition of hepatic fibrogenesis. It is reported to act on the Nrf2/Keap1/HO-1, TLR4/NF-κB, NLRP3 inflammasome, AMPK-associated metabolic pathways and TGF-β1/Smad signaling, making it a potential therapeutic molecule targeting multiple targets. Experimental studies have shown promising hepatoprotective activity in various models of metabolic dysfunction-associated steatotic liver disease, steatohepatitis, alcohol-associated liver injury, hepatotoxicity induced by drugs, and hepatic fibrosis. Based on these results, andrographolide could have an impact on multiple pathological mechanisms involved in the onset and development of liver diseases. However, most of the evidence comes from cell and animal research, and evidence of efficacy in humans is also lacking. Moreover, inadequate aqueous solubility, variable bioavailability following oral administration, metabolic transformation and lack of long-term safety data are all major challenges in pharmaceutical development. The novel technologies like nanotechnology based drug delivery system, modification of structure and targeted delivery system provide opportunities to enhance the pharmacokinetic property and therapeutic activity of andrographolide. These strategies, however, necessitate the strict validation under standardized experimental models, comparative pharmacological studies and extensive toxicological evaluation. Further clinical research should focus on appropriate dosing, formulation standardisation, PK evaluation and clinically-relevant hepatic outcomes. The overall opinion is that A. paniculata and andrographolide are potential candidates for drug discovery and development for hepatoprotection instead of conventional drugs for liver disease. Incorporating molecular pharmacology, advanced formulation development, and carefully designed clinical research will play a significant role in establishing the therapeutic potential and safety of these compounds and supporting the future development of safe and effective evidence-based therapies for liver disorders.

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